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This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan, and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV , respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species, with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a range similar to human parameters and good predictions from respective hepatocyte parameters (with 2.7- and 3.8-fold bias on average, respectively). The use of cross-species empirical scaling factors (determined from cynomolgus monkey data either as the data set average or individual drug values) improved prediction (less bias, better concordance) of human hepatic clearance from human hepatocyte data alone. In vitro intracellular binding in hepatocytes also correlated well between species. It is concluded that the minimal species differences observed for the current data set between cynomolgus monkey and human hepatocyte uptake, both in vitro and in vivo, support future use of this preclinical model to delineate drug hepatic uptake and enable prediction of human in vivo intrinsic hepatic clearance.

In vitro-in vivo extrapolation (IVIVE) to predict human hepatic clearance including metabolism and transport requires extensive experimental resources. In addition, there may be technical challenges to measure low clearance values. Therefore, prospective identification of rate-determining step(s) in hepatic clearance through application of the Extended Clearance Classification System (ECCS) could be beneficial for optimal compound characterization. IVIVE for hepatic intrinsic clearance (CLint,h) prediction is conducted for a set of 36 marketed drugs with low-to-high in vivo clearance, which are substrates of metabolic enzymes and active uptake transporters in the liver. The compounds were assigned to the ECCS classes and CLint,h, estimated with HepatoPac® (a micro-patterned hepatocyte co-culture system), was compared to values calculated based on suspended hepatocyte incubates. A P app threshold (apical to basal) of 50 nm/s in LLC-PK1 cells proved optimal for ECCS classification. A reasonable performance of the IVIVE for compounds across multiple classes using HepatoPac® was achieved (with 2-to 3-fold error), except for substrates of uptake transporters (class 3b), where scaling of uptake clearance using plated hepatocytes is more appropriate. Irrespective of the ECCS assignment, metabolic clearance can be estimated well using HepatoPac®. The validation and approach elaborated in the present study can result in proposed decision trees for the selection of the optimal in vitro assays guided by ECCS class assignment, to support compound optimization and candidate selection. Significant Statement Characterization of the rate-determining step(s) in hepatic elimination could be on the critical path of compound optimization during drug discovery. This study demonstrated that HepatoPac® and plated hepatocytes are suitable tools for the estimation of metabolic and active uptake clearance, respectively, for a larger set of marketed drugs, supporting a This article has not been copyedited and formatted. The final version may differ from this version.